Traditional flow cytometry allows for fluorescently labeled live cells, fixed cells, beads, or objects to be individually distinguished and separated using cytometric sorting technology based on their fluorescent and light scatter characteristics. This approach is particularly advantageous because it allows for further functional or analytical characterization of individually purified cells on a phenotypic basis.
An essential phenotype measured in most cell analysis experiments is cell viability, as dead cells can easily distort the analyzed phenotypes. Cell viability is typically determined by molecules that cannot pass the intact membrane of a live cell, but which enter the cytoplasm and nucleus of dead cells. Examples include propidium iodide and ethidium monoazide, which intercalate or covalently bind to DNA. Alternatively, fluorescently labeled affinity binders specific for cell death markers, such as cleaved Caspase 3, cleaved Parp or Annexin V can be used for this purpose. As a result live and dead cells can be easily distinguished based on the fluorescence intensity of the fluorophore used for the viability stain.
Elemental mass spectrometry-based flow cytometry (mass cytometry) is a recently developed method to characterize single cells or particles via the replacement of fluorochrome-labeled binding reagents with elemental metal isotope-labeled binding reagents. Because there are many stable metal isotopes available, and little overlap between measurement channels, dozens of molecules (parameters) can be readily measured. An example of a mass cytometer used to read the metal tags is an inductively-coupled plasma mass spectrometer (ICP-MS), which in its current configuration allows analysis of up to 100 isotopes from the mass 100-200 and therefore molecules simultaneously2. In a typical workflow (similar to fluorescence based cytometry), cells are first incubated with antibodies/affinity binders conjugated to pure isotopes and subsequently the cell suspension is injected as a single cell stream into the mass cytometer. Single cell droplets are generated via nebulization and are carried by an argon gas stream into a ˜7500 degrees Kelvin plasma where each single cell is completely atomized and ionized. Thereby generated metal ions are then directed into a time-of-flight (TOF) mass spectrometer and the mass over charge ratio and number of metal ions is measured per cell and thereby the abundance of the target epitope/molecules.
The value of this mass cytometry-based technique, including the routine measurement of up to 33 parameters, has recently been documented. One limitation, however, is that no robust method for an accurate discrimination of live and dead cells in a given sample exists. Conventional methods developed for flow cytometry methods rely on metal labeled DNA intercalators, however, as these compounds are not covalently bound to the cell, they are easily washed out in subsequent sample processing steps, making the reliable determination of a cells live/dead status very difficult.
Consequently, many analyses performed with mass cytometry fall short of yielding an accurate picture of a phenotypic signature; and it is not possible to determine the impact of the dead cells on the performed experiment. For example, dead cells can exhibit false-negative or false-positive staining for various surface and intracellular targets. A viability assay is needed to positively identify these dead and resolve the heterogeneous staining. Additionally, quantification of dead cells is helpful in measuring the efficacy of in vitro drug treatments meant to kill cells (i.e. chemotherapeutic agents). Therefore, there is great value in a method that overcomes the lack of a live/dead cellular viability assay. The present invention addresses this need.
Publications
Fienberg et al. (2012) Cytometry A 81(6):467-75, entitled “A platinum-based covalent viability reagent for single-cell mass cytometry.” Majonis et al. (2011) Biomacromolecules 12(11):3997-4010, entitled “Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.” Bendall et al. (2011) Science 332(6030):687-96, entitled “Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.” Ornatsky et al. (2008) J Anal At Spectrom 23(4):463-469, entitled “Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.” Rieger et al. (2010) J Immunol Methods. 358(1-2):81-92, entitled “Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.” Stocks (2004) Cytometry A. 61(2):189-95, entitled “Mechanism and use of the commercially available viability stain, BacLight.” Berber et al. (1999) Cytometry. 36(4):349-54, entitled “Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.”